Galectin-1/ galectin-3 chimeras and multivalent proteins

This application is a national stage filing under 35 U.S.C. 371 of International Patent Application Serial No. PCT/US2020/020532, filed Feb. 28, 2020, which claims the benefit under 35 USC 119(e) of the filing date of U.S. Patent Application Ser. No. 62/811,771, filed Feb. 28, 2019. The contents of the above-referenced applications are hereby incorporated herein in their entirety by reference.

This invention was made with government support under Grant Number EB019684 awarded by the National Institutes of Health. The government has certain rights in the invention.

Galectins, a 15-member family of soluble carbohydrate-binding proteins, are interesting as therapeutic targets for immunotherapy and immunomodulation due to their role as extracellular signals that regulate innate and adaptive immune cell phenotype and function. However, different galectins can have redundant, synergistic, and/or antagonistic signaling activity in normal immunological responses, such as resolution of inflammation and induction of antigen-specific tolerance. In addition, certain galectins can be used to promote progression of immune-pathologies, such as tumor immune privilege, metastasis, and viral infection, while others can inhibit these processes.

Although glycobiology research was first recognized in the early 19century, researchers are just now beginning to understand the complex role of lectins and glycans within the immune system.Galectins are a family of soluble, beta-galactoside-binding lectins that regulate the phenotype and function of various immune cells during the initiation and resolution of immune responses.For example, galectin-1 (G1) and galectin-3 (G3) are mediators of tumor immunosuppression and fetal-maternal tolerance, regulate autoimmune disease progression, can enhance or inhibit viral infection, and induce pro-inflammatory responses during osteoarthritis.

Extracellular G1 and G3 can act on innate immune cells, including monocytes, macrophages, neutrophils, and dendritic cells, yet it is their influence on T cells that receives the most attention for immunotherapy.Extracellular G1 and G3 can induce T cell apoptosis, regulate antigen-specific T cell activation, and alter T cell cytokine secretion.Despite sharing binding affinity for beta-galactoside glycans, though, G1 and G3 often evoke these changes in T cells by recognizing different cell surface glycoproteins, suggesting they function through different signalling pathways.For example, although both G1 and G3 bind to CD45, only G1 induces CD45 clustering to trigger T cell apoptosis.Both G1 and G3 have been shown to interact with CD7, yet G3 binding to CD7 does not trigger T cell death.G1 can bind to CD2 and CD3, whereas G3 binding to these glycoproteins has not been observed.G1 can also selectively induce death of T helper (Th)1 and Th17 cells, yet has no effect on Th2 and regulatory T cells, due to polarization-induced changes in the T cell surface glycosylation profile.Similarly, G3 preferentially kills double-negative thymocytes, but not double-positive thymocytes, again due to alterations in the surface glycosylation profile of cells as they mature or polarize.Interestingly, a combination of G1 and G3 did not have an additive or synergistic effect on T cell apoptosis, but rather elicited a similar extent of cell death as either galectin alone, possibly due to competitive interactions with CD45 or other T cell surface glycoproteins.Beyond apoptosis, G1 has been shown to stimulate antigen-specific T cell responses while G3 antagonized these responses, yet in other contexts both G1 and G3 can regulate T cell receptor clustering and signaling.Finally, G1 and G3 also have differing effects on T cell cytokine secretion, with low concentration G3 inducing IL-2 secretion, while G1 upregulates IL-10 expression by Th17 cells.

In light of these diverse effects on T cells, exogenous galectins and engineered variants have been evaluated as therapeutics to regulate adaptive immunity in various contexts.Recombinant G1 has received the most attention to date, and has demonstrated efficacy in rodent models of Crohn's disease, multiple sclerosis, myasthenia gravis, rheumatoid arthritis, graft vs. host disease, autoimmune uveitis, and T cell mediated hepatitis.A key aspect of G1 and G3 extracellular activity is their non-covalent self-association into quaternary structures with multiple carbohydrate-recognition domains (CRDs). For example, G1 associates into homodimers at relatively high (μM) concentrations.To stabilize its activity in dilute conditions, G1 dimers formed via a polypeptide linker, a synthetic polymer, a leucine zipper coiled-coil forming peptide, or an IgG Fc domain were engineered.Often, these engineered G1 variants demonstrate a minimum effective dose that is approximately 10-fold lower than that of the wild-type protein. In contrast, G3 is unique among galectins in that it assembles into higher-ordered oligomers (≥2 G3 molecules) upon glycan binding via interactions involving its N-terminal domain.No engineered G3 oligomers with improved activity have been reported. Rather, G3 inhibitors have been developed by treating the wild-type protein with collagenase, which cleaves the N-terminal domain without disrupting the CRD.Likewise, it was recently reported G3 fusion proteins that bind glycans yet lack activity for inducing T cell agglutination, apoptosis, and cytokine secretion because they cannot oligomerize.Together, these examples demonstrate the potential of protein engineering approaches to manipulate G1 and G3 immunomodulatory activity by altering their oligomerization.Despite these efforts, though, existing engineered G1 and G3 variants still have minimum effective doses that are in the low μM range (0.5-5 μM), which is impractical for many therapeutic applications. Described herein, is a way to further decrease the effective dose of G1 and G3 can be to combine them into chimeric multivalent assemblies.

In an aspect, the disclosure relates to a Galectin-1/Galectin-3 (Gal-1/Gal-3) dimer comprising, two monomer polypeptides, wherein each monomer polypeptide comprises, a Galectin-1 (Gal-1) polypeptide; a Galectin-3 (Gal-3) polypeptide; and an alpha helix coil, wherein the alpha helix coil is fused between the Gal-1 polypeptide and the Gal-3 polypeptide, wherein the two monomer polypeptides associate with each other via dimerization at the alpha helix coils to form the Gal-1/Gal-3 dimer.

In some embodiments, one or both of the monomer polypeptides has a sequence that is about 90% to about 100% identical to SEQ ID NO: 3. In some embodiments, one or both of the monomer polypeptides comprises the sequence of SEQ ID NO: 3. In some embodiments, one or both of the monomer polypeptides comprises the sequence of SEQ ID NO: 3 with at least one amino acid substitutions as compared to SEQ ID NO: 3. In some embodiments, the Gal-1 polypeptide has a sequence that is about 80% to about 100% identical to SEQ ID NO: 1. In some embodiments, the Gal-1 polypeptides comprises the sequence of SEQ ID NO: 1. In some embodiments, the Gal-1 polypeptides comprises the sequence of SEQ ID NO: 1 with at least one amino acid substitutions as compared to SEQ ID NO: 1. In some embodiments, the Gal-3 polypeptide has a sequence that is about 80% to about 100% identical to SEQ ID NO: 4. In some embodiments, the Gal-3 polypeptides comprises the sequence of SEQ ID NO: 4. In some embodiments, the Gal-3 polypeptides comprises the sequence of SEQ ID NO: 4 with at least one amino acid substitutions as compared to SEQ ID NO: 4. In some embodiments, the Gal-3 polypeptide has a sequence that is about 80% to about 100% identical to SEQ ID NO: 4 less the underlined and bolded histidine tag (i.e., the sequence as set forth omitting the underlined and bolded amino acid residues). In some embodiments, the Gal-3 polypeptides comprises the sequence of SEQ ID NO: 4 less the underlined and bolded histidine tag (i.e., the sequence as set forth omitting the underlined and bolded amino acid residues). In some embodiments, the Gal-3 polypeptides comprises the sequence of SEQ ID NO: 4 less the underlined and bolded histidine tag (i.e., the sequence as set forth omitting the underlined and bolded amino acid residues) with at least one amino acid substitutions as compared to SEQ ID NO: 4 less the underlined and bolded histidine tag (i.e., the sequence as set forth omitting the underlined and bolded amino acid residues).

In some embodiments, the alpha helix coil has a sequence that is about 80% to about 100% identical to any one of SEQ ID NO: 5. In some embodiments, the alpha helix coil comprises the sequence of SEQ ID NO: 5. In some embodiments, the alpha helix coil comprises the sequence of SEQ ID NO: 5 with at least one amino acid substitutions as compared to SEQ ID NO: 5.

In an aspect, the disclosure relates to a Galectin-1/Galectin-3 (Gal-1/Gal-3) polypeptide capable of dimerizing comprising, a Galectin-1 (Gal-1) polypeptide; a Galectin-3 (Gal-3) polypeptide; and an alpha helix coil, wherein the alpha helix coil is fused between the Gal-1 polypeptide and the Gal-3 polypeptide.

In some embodiments, the Gal-1/Gal-3 polypeptide has a sequence that is about 90% to about 100% identical to SEQ ID NO: 3. In some embodiments, the Gal-1/Gal-3 polypeptide comprises the sequence of SEQ ID NO: 3. In some embodiments, the Gal-1/Gal-3 polypeptide comprises the sequence of SEQ ID NO: 3 with at least one amino acid substitutions as compared to SEQ ID NO: 3. In some embodiments, the Gal-1 polypeptide has a sequence that is about 80% to about 100% identical to SEQ ID NO: 1. In some embodiments, the Gal-1 polypeptides comprises the sequence of SEQ ID NO: 1. In some embodiments, the Gal-1 polypeptides comprises the sequence of SEQ ID NO: 1 with at least one amino acid substitutions as compared to SEQ ID NO: 1. In some embodiments, the Gal-3 polypeptide has a sequence that is about 80% to about 100% identical to SEQ ID NO: 4. In some embodiments, the Gal-3 polypeptides comprises the sequence of SEQ ID NO: 4. In some embodiments, the Gal-3 polypeptides comprises the sequence of SEQ ID NO: 4 with at least one amino acid substitutions as compared to SEQ ID NO: 4. In some embodiments, the Gal-3 polypeptide has a sequence that is about 80% to about 100% identical to SEQ ID NO: 4 less the underlined and bolded histidine tag (i.e., the sequence as set forth omitting the underlined and bolded amino acid residues). In some embodiments, the Gal-3 polypeptides comprises the sequence of SEQ ID NO: 4 less the underlined and bolded histidine tag (i.e., the sequence as set forth omitting the underlined and bolded amino acid residues). In some embodiments, the Gal-3 polypeptides comprises the sequence of SEQ ID NO: 4 less the underlined and bolded histidine tag (i.e., the sequence as set forth omitting the underlined and bolded amino acid residues) with at least one amino acid substitutions as compared to SEQ ID NO: 4 less the underlined and bolded histidine tag (i.e., the sequence as set forth omitting the underlined and bolded amino acid residues).

In some embodiments, the alpha helix coil has a sequence that is about 80% to about 100% identical to SEQ ID NO: 5. In some embodiments, the alpha helix coil comprises the sequence of SEQ ID NO: 5. In some embodiments, the alpha helix coil comprises the sequence of SEQ ID NO: 5 with at least one amino acid substitutions as compared to SEQ ID NO: 5.

In an aspect, the disclosure relates to a Galectin-1/Galectin-3 (Gal-1/Gal-3) polynucleotide comprising, a polynucleotide that encodes a Galectin-1 (Gal-1) polypeptide; a polynucleotide that encodes a Galectin-3 (Gal-3) polypeptide; and a polynucleotide that encodes an alpha helix coil, wherein the polynucleotide that encodes the alpha helix coil is fused in-frame between the polynucleotide that encodes the Gal-1 polypeptide and the polynucleotide that encodes the Gal-3 polypeptide.

In some embodiments, the polynucleotide encodes a Gal-1 polypeptide comprising a sequence that is about 80% to about 100% identical to SEQ ID NO: 1. In some embodiments, the polynucleotide encodes a Gal-1 polypeptide comprising the sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide encodes a Gal-1 polypeptide comprising the sequence of SEQ ID NO: 1 with at least one amino acid substitutions as compared to SEQ ID NO: 1. In some embodiments, the polynucleotide encodes a Gal-3 polypeptide comprising a sequence that is about 80% to about 100% identical to SEQ ID NO: 4. In some embodiments, the polynucleotide encodes a Gal-3 polypeptide comprising the sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide encodes a Gal-3 polypeptide comprising the sequence of SEQ ID NO: 4 with at least one amino acid substitutions as compared to SEQ ID NO: 4. In some embodiments, the polynucleotide encodes a Gal-3 polypeptide comprising a sequence that is about 80% to about 100% identical to SEQ ID NO: 4 less the underlined and bolded histidine tag (i.e., the sequence as set forth omitting the underlined and bolded amino acid residues). In some embodiments, the polynucleotide encodes a Gal-3 polypeptide comprising the sequence of SEQ ID NO: 4 less the underlined and bolded histidine tag (i.e., the sequence as set forth omitting the underlined and bolded amino acid residues). In some embodiments, the polynucleotide encodes a Gal-3 polypeptide comprising the sequence of SEQ ID NO: 4 less the underlined and bolded histidine tag (i.e., the sequence as set forth omitting the underlined and bolded amino acid residues) with at least one amino acid substitutions as compared to SEQ ID NO: 4 less the underlined and bolded histidine tag (i.e., the sequence as set forth omitting the underlined and bolded amino acid residues).

In some embodiments, the polynucleotide encodes an alpha helix coil comprising a sequence that is about 80% to about 100% identical to SEQ ID NO: 5. In some embodiments, the polynucleotide encodes an alpha helix coil comprising the sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide encodes an alpha helix coil comprising the sequence of SEQ ID NO: 5 with at least one amino acid substitutions as compared to SEQ ID NO: 5.

In an aspect, the disclosure relates to a vector comprising, any of the Gal-1/Gal-3 polynucleotides as described herein.

In some embodiments, the Gal-1/Gal-3 polynucleotide is operatively linked to a regulatory polynucleotide.

In an aspect, the disclosure relates to a bacterial cell comprising, any of the polynucleotide sequences as described herein or any of the vectors as described herein.

In an aspect, the disclosure relates to a pharmaceutical formulation comprising, any of the Galectin-1/Galectin-3 (Gal-1/Gal-3) dimers as described herein; and a pharmaceutically acceptable carrier.

In an aspect, the disclosure relates a pharmaceutical formulation comprises a plurality of any of the Galectin-1/Galectin-3 (Gal-1/Gal-3) polypeptides capable of dimerizing as described herein; and a pharmaceutical formulation thereof.

In an aspect, the disclosure relates to a method comprising: administering any of the Galectin-1/Galectin-3 (Gal-1/Gal-3) dimers as described herein or a pharmaceutical formulation thereof to a subject in need thereof.

In some embodiments, the subject in need thereof has inflammatory disease. In some embodiments, the subject in need thereof has an autoimmune disease. In some embodiments, the subject in need thereof has arthritis. In some embodiments, the subject in need thereof has osteoarthritis. In some embodiments, the subject in need thereof has rheumatoid arthritis. In some embodiments, the subject in need thereof has diabetes. In some embodiments, the subject in need thereof has type I diabetes. In some embodiments, the subject in need thereof has multiple sclerosis. In some embodiments, the subject in need thereof has graft versus host disease. In some embodiments, the subject in need thereof has colitis. In some embodiments, the subject in need thereof has uveitis. In some embodiments, the subject in need thereof is rejecting a transplanted organ or tissue.

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are cited to disclose and describe the methods and/or materials in connection with which the publications are cited. All such publications and patents are herein incorporated by references as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and/or materials described in the cited publications and patents and does not extend to any lexicographical definitions from the cited publications and patents. Any lexicographical definition in the publications and patents cited that is not also expressly repeated in the instant application should not be treated as such and should not be read as defining any terms appearing in the accompanying claims. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Where a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g., ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y′, and ‘less than z’ Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y′, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

As used herein, “about,” “approximately,” “substantially,” and the like, when used in connection with a numerical variable, can generally refers to the value of the variable and to all values of the variable that are within the experimental error (e.g., within the 95% confidence interval for the mean) or within +/−10% of the indicated value, whichever is greater. As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” can mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of molecular biology, microbiology, nanotechnology, organic chemistry, biochemistry, botany and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible unless the context clearly dictates otherwise.

As used herein, “active agent” or “active ingredient” refers to a substance, compound, or molecule, which is biologically active or otherwise, induces a biological or physiological effect on a subject to which it is administered to. In other words, “active agent” or “active ingredient” refers to a component or components of a composition to which the whole or part of the effect of the composition is attributed.

As used herein, “additive effect” refers to an effect arising between two or more molecules, compounds, substances, factors, or compositions that is equal to or the same as the sum of their individual effects.

As used herein, “administering” refers to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intraosseous, intraocular, intracranial, intraperitoneal, intralesional, intranasal, intracardiac, intraarticular, intracavernous, intrathecal, intravireal, intracerebral, and intracerebroventricular, intratympanic, intracochlear, rectal, vaginal, by inhalation, by catheters, stents or via an implanted reservoir or other device that administers, either actively or passively (e.g., by diffusion) a composition the perivascular space and adventitia. For example a medical device such as a stent can contain a composition or formulation disposed on its surface, which can then dissolve or be otherwise distributed to the surrounding tissue and cells. The term “parenteral” can include subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intracisternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.

As used herein, “agent” refers to any substance, compound, molecule, and the like, which can be biologically active or otherwise can induce a biological and/or physiological effect on a subject to which it is administered to. An agent can be a primary active agent, or in other words, the component(s) of a composition to which the whole or part of the effect of the composition is attributed. An agent can be a secondary agent, or in other words, the component(s) of a composition to which an additional part and/or other effect of the composition is attributed.

As used herein, “alpha helix coil,” or “a helix” as it may also be referred to, refers to a secondary structure of a protein (e.g., peptide of more than one amino acid residue), which consists of a peptide chain coiled into a right-handed spiral conformation, which conformation is stabilized by hydrogen bonds between the NH and CO groups in the backbone of the peptide.

As used herein, “amphiphilic” can refer to a molecule combining hydrophilic and lipophilic (hydrophobic) properties.

As used herein, “antibody” can refer to a glycoprotein containing at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region and a light chain constant region. The VH and VL regions retain the binding specificity to the antigen and can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR). The CDRs are interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four framework regions, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

As used herein, “anti-infective” refers to compounds or molecules that can either kill an infectious agent or inhibit it from spreading. Anti-infectives include, but are not limited to, antibiotics, antibacterials, antifungals, antivirals, and antiprotozoans.

As used herein, “aptamer” can refer to single-stranded DNA or RNA molecules that can bind to pre-selected targets including proteins with high affinity and specificity. Their specificity and characteristics are not directly determined by their primary sequence, but instead by their tertiary structure.

The term “biocompatible”, as used herein, refers to a material that along with any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause any significant adverse effects to the recipient. Generally speaking, biocompatible materials are materials that do not elicit a significant inflammatory or immune response when administered to a patient.

The term “biodegradable” as used herein, generally refers to a material that will degrade or erode under physiologic conditions to smaller units or chemical species that are capable of being metabolized, eliminated, or excreted by the subject. The degradation time is a function of composition and morphology. Degradation times can be from hours to weeks.

As used herein, “cDNA” refers to a DNA sequence that is complementary to a RNA transcript in a cell. It is a man-made molecule. Typically, cDNA is made in vitro by an enzyme called reverse-transcriptase using RNA transcripts as templates.

As used herein, “chemotherapeutic agent” or “chemotherapeutic” refers to a therapeutic agent utilized to prevent or treat cancer.

As used herein, “concentrated” can refer to a molecule or population thereof, including but not limited to a polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, that is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than that of its naturally occurring counterpart.

As used herein, “control” can refer to an alternative subject or sample used in an experiment for comparison purpose and included to minimize or distinguish the effect of variables other than an independent variable.

As used herein with reference to the relationship between DNA, cDNA, cRNA, RNA, protein/peptides, and the like “corresponding to” or “encoding” (used interchangeably herein) refers to the underlying biological relationship between these different molecules. As such, one of skill in the art would understand that operatively “corresponding to” can direct them to determine the possible underlying and/or resulting sequences of other molecules given the sequence of any other molecule which has a similar biological relationship with these molecules. For example, from a DNA sequence an RNA sequence can be determined and from an RNA sequence a cDNA sequence can be determined.

As used herein, “deoxyribonucleic acid (DNA)” and “ribonucleic acid (RNA)” can generally refer to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. RNA can be in the form of non-coding RNA such as tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), anti-sense RNA, RNAi (RNA interference construct), siRNA (short interfering RNA), microRNA (miRNA), or ribozymes, aptamers, guide RNA (gRNA) or coding mRNA (messenger RNA).